The transforming growth factor-beta (TGF-.beta.) superfamily of cytokines regulate a diverse array of physiologic functions including cell proliferation and growth, cell migration, differentiation, development and apoptosis. This large family includes the TGF-.beta.s, activins, and bone-morphogenic proteins (BMPs) and each subgroup initiates a unique signaling cascade activated by ligand-induced serine/threonine kinase receptor complex formation (Wrana, Miner. Electrolyte Metab., 1998, 24, 120-130). These complexes, once formed, recruit and phosphorylate members of a family of cytosolic proteins, known as Smads. Smads exist as monomers in unstimulated cells but homo- or heterodimerize and translocate to the nucleus activating target gene transcription upon ligand binding. Smads, therefore, connect the pathway of TGF-.beta. signaling from the cell membrane to the nucleus.
To date, nine vertebrate Smads have been identified and these have been divided into subgroups based on their functional role in various pathways. Smad1, 5, and MADH6, which is 80% homologous to Smad1, all mediate signal transduction from BMPs while Smad2 and 3 mediate signal transduction from TGF-.beta.s and activins. Collectively, these Smads are known as the pathway-restricted Smads and can form homo or heterodimers. Smad4 has been shown to be a shared hetero-oligomerization partner to the pathway-restricted Smads and is known as the common mediator. The last two members of the family, Smad6 and 7, act to inhibit the Smad signaling cascades often by forming unproductive dimers with other Smads and are therefore classified as antagonistic Smads (Heldin et al., Nature, 1997, 390, 465-471; Kretzschmar and Massague, Curr. Opin. Genet. Dev., 1998, 8, 103-111).
Smad2 (also known as MADH2, MADR2, hMAD2 and JV18-1) is a member of a subgroup of Smad family transcription factors which are regulated by TGF-.beta. and activins. Upon ligand binding Smad2 becomes phosphorylated and associates with Smad3. This complex then associates with Smad4 and translocates to the nucleus where it effects transcription of target genes. It has been demonstrated that the phosphorylation of Smad2 is necessary for the association with Smad4 (Souchelnytskyi et al., J. Biol. Chem., 1997, 272, 28107-28115) and that Smad2 and Smad4 interact with CREB binding protein, an essential component of the mammalian transcription apparatus (Topper et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 9506-9511).
The Smad2 gene is located on chromosome 18q21, a region that frequently undergoes allelic loss in many cancers. A missense somatic mutation and a 9-bp in-frame deletion were detected in the highly conserved region of JV18-1 among 57 lung cancer specimens taken directly from patients (Uchida et al., Cancer Res., 1996, 56, 5583-5585). In addition, missense and nonsense mutations of the Smad2 gene have also been found in 6-17% of colorectal carcinoma cell lines and primary tumors (Eppert et al., Cell, 1996, 86, 543-552).
In normal cells, Smad2 acts to transmit signals from TGF-.beta. and the activins. It has also been shown to mediate cross-talk between receptor tyrosine kinase pathways and receptor serine/threonine kinase pathways by acting as a positive effector in the EGF and HGF signaling cascades (de Caestecker et al., Genes Dev., 1998, 12, 1587-1592).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Smad2 and to date, strategies aimed at inhibiting Smad2 function have involved the use of dominant-negative mutants of Smad2, gene knock-outs in mice and antisense oligonucleotides designed against Smad2.
Studies of mice lacking the Smad2 gene showed that Smad2 is necessary for embryonic mesoderm formation and the establishment of anterior-posterior polarity (Waldrip et al., Cell, 1998, 92, 797-808). Analysis of mice lacking one copy of the gene found that developmental changes depended on the amount of Smad2 activity and that a defective phenotype was apparent when both of the Smad2 genes are inactivated (Nomura and Li, Nature, 1998, 393, 786-790).
Antisense oligonucleotides designed against Smad2 were used in studies of lung morphogenesis to show that Smad2 negatively regulates lung organogenesis. In these studies, it was demonstrated that treatment of embryonic mouse lung cultures with Smad2 antisense oligonucleotides resulted in increased lung branching morphogenesis (Zhao et al., Dev. Biol., 1998, 194, 182-195).
In light of the limited strategies available for targeting Smad2 function, there remains a long felt need for additional agents capable of effectively inhibiting Smad2. Therefore, antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of Smad2 expression.